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1 微生物学通报 DEC 20, 2009, 36(2): 832~837 Microbiology 研究报告 2009 by Institute of Microbiology, CAS 拮抗放线菌 S24 的鉴定及其对黄曲霉的抑制作用周启升孙长坡 2 张楠 3 仇念全曹宁宁张本峰刘训理 * (. 2708) ( ) ( ) 摘要 : 以黄曲霉 (Aspergillus flavus) 为靶标, 从泰山土壤中分离获得一株对黄曲霉赭曲霉黑曲霉等粮食和饲料中常见的曲霉菌有高效拮抗活性的放线菌 S24 根据其形态特征培养特征理化性质细胞壁组份及 6S rrna 序列分析, 初步判定该菌株为链霉菌属中的白网链霉菌 (Streptomyces albireticuli) 的近似种 ; 该菌株抗菌谱广, 胞外抗菌物质对热稳定, 00 C 加热 00 min 抗菌活性无明显变化 ; 96 孔板法测得其胞外抗菌物质粗提物对黄曲霉的最小抑 / 杀菌浓度 (MIC/MFC) 分别为 9.53 µg/ml 和 µg/ml 关键词 : 黄曲霉, 白网链霉菌, 鉴定, 最小抑 / 杀菌浓度 Identification of Actinomycete Strain S24 and Its Antagonistic Effects on Aspergillus flavus ZHOU Qi-Sheng SUN Chang-Po 2 ZHANG Nan 3 QIU Nian-Quan CAO Ning-Ning ZHANG Ben-Feng LIU Xun-Li * (. College of Forestry, Shandong Agricultural University, Taian, Shandong 2708, China) (2. Academy of State Administration of Grain, Beijing 00037, China) (3. College of Life Science, Shandong Agricultural University, Taian, Shandong 2708, China) Abstract: Taking Aspergillus flavus as the target, Actinomyces S24 isolated from Mountain Tai soil showed strong antagonistic activity against Aspergillus flavus, Aspergillus niger, Asperegillus alutacells and the other common Aspergillus in food and feed. Based on its morphologic, culture, physiological and biochemical characteristics, chemotaxonomy as well as 6S rrna sequence analysis, strain S24 was placed within the genus Streptomyces and identified as a similar species with Streptomyces albireticuli, which had a broad inhibition spectrum and its fermentation characteristics were outstanding. The antibacterial substance was stable. Deposed for 00 min at 00 C, there was no obvious influence on its antibacterial activity. Assay using 96-well plate illustrated that the minimum inhibitor concentration (MIC) and minimum antifungal concentration (MFC) of extracellular crude antibacterial extracts against A. flavus were 9.53 µg/ml and µg/ml, respectively. Keywords: Aspergillus flavus, Streptomyces albireticuli, Identification, MIC/MFC * 通讯作者 :Tel: ; : xlliu@sdau.edu.cn 收稿日期 : ; 接受日期 :

2 : S (Aspergillus flavus),,, [], [2 4], (Aflatoxins),, 993 (IARC) [5], [6],, Vargas [7] (Larrea tridentata), ; Sanchez [8] Agave asperrima Agave striata, ; Klich [9] ; Yadav [0], (Escherichia coli) ; [],, 3.2 µg/ml; Roy [2] 6, 5 mm~9 mm,, S24, 6S rrna, 材料与方法. 材料.. 供试菌株 : S24 ; : (Aspergillus flavus CGMCC3.2890), (Aspergillus niger CGMCC3.6478), (Asperegillus alutacells CGMCC3.409), (Aspergillus fumigatus CGMCC3.3552),..2 培养基 : : 0.0 g, FeSO 4 7H 2 O 0.0 g, KNO 3.0 g, 5.0 g, MgSO 4 7H 2 O 0.5 g, NaCl 0.5 g, ph 7.2~7.4, 000 ml PDA : 200 g, 5.0 g, 20 g, 5 g, (NH 4 ) 2 SO 4.0 g, MgSO 4.0 g, KH 2 PO g, CaCO g, ph 6.8~7.2, 000 ml : 200 g, 20 g, 5 g, (NH 4 ) 2 SO 4.0 g, MgSO 4.0 g, KH 2 PO g, ph 6.8~7.2, 000 ml S24 : 6.0 g, 2.5 g, 0 g, 26 g,.0 g, NaCl 7.5 g, (NH 4 ) 2 SO g, ph, 000 ml..3 主要试剂和仪器 : DNA PCR ; ; SPX ; Olympus ; Bio-Rad PTC-200 PCR Lab Assistant Gel 3000 Series ; HZQ-F60 ; Heraeus.2 方法.2. 菌株的鉴定 : : S24, 28 C 7 d~4 d,, :, 28 C 7 d~4 d [3] : (TCL) [4] 6S rrna : DNA 6S rrna [5,6]6S rrna 6S rrna, 6Sf: AGA GTTTGATCCTGGCTCAG; 6Sr: ACG GCTACCTTGTTACGACT,.6 kb, PCR %, DL2000 Marker U-GENE,,

3 834 微生物学通报 2009, Vol.36, No.2 GenBank Blast,, Mega4.0 Neighbor-Joining.2.2 抗菌谱的测定 :, 2 cm S24, 28 C 96 h,,,.2.3 抗菌物质粗提物的制备 : 0 5 Pa 20 min, S24 ( 8 mm) 50 ml/250 ml, 200 r/min, 30 C 48 h 0 4 Pa 0 min, 4200 r/min, 30 min,,.2.4 抗菌物质效价的生物测定 : :, 50 C~55 C,, CFU/mL ~4 0 4 CFU/mL, 90 mm, 20 ml/,, ( 6 mm ± 0. mm, 0 mm ± 0. mm, 8 mm ± 0. mm)6 250 µl, 3 25 C 8 h : 00 µg/ml 50 µg/ml 200 µg/ml 250 µg/ml 300 µg/ml 400 µg/ml 500 µg/ml,,,, R 2 = ,, 图杯碟法测定效价工作曲线 Fig. The curve for working of portency mensuration by means of cylinde-plate method : Y = 0 (x+0.93)/0.797 n Y: (µg/ml); x: (0.5 mm < x < 8.0 mm); n:.2.5 抗菌物质对温度的敏感性试验 : 5000 µg/ml, 00 C 0 min 20 min 40 min 60 min 80 min 00 min 0 5 Pa 5 min,.2.6 抗菌物质对黄曲霉最小抑 / 杀菌浓度 (MIC 和 MFC) 的测定 : : 96, 50 µl RPMI-640, 2~ 75 µl RPMI-640 2, : 2500 µg/ml 250 µg/ml 625 µg/ml 32.5 µg/ml µg/ml 78.3 µg/ml µg/ml 9.53 µg/ml 9.77 µg/ml 4.88 µg/ml 2~2 0 4 CFU/mL ~2 0 4 CFU/mL 75 µl, 2 75 µl RPMI-640 MIC MFC : (NCCLS) 2 结果与分析 2. S24 菌株的形态学观察结果 S24,,, ; ;,,, 2.2 S24 菌株的培养特征和生理生化特征 S24 8 ( ), ; ( 2), 5 C~37 C, 28 C; ph 4~2,,,, 2.3 S24 菌株的细胞壁化学成分分析结果 S24,, C; LL- (LL-DAP)

: S24 835 Medium 表菌株 S24 的培养特征 Table Cultural characteristics of strain S24 Color of aerial mycelium Color of substrate mycelium Color of soluble pigments Mycelial growth Gauze No.

4 : S Medium 表菌株 S24 的培养特征 Table Cultural characteristics of strain S24 Color of aerial mycelium Color of substrate mycelium Color of soluble pigments Mycelial growth Gauze No. sagars Hoariness Mustard Black +++ Inorganic salt agars Hoariness Bamboo palm None ++++ Salt-starch agar Breast white Chorion yellow None + PDA agars Bamboo palm Sepia ash Coffee ++ Glucose/Asparagine agars Lichee white Ivoryyellow None + Glycerol/Asparagine agar Dull green Fleet pine mist None ++ Czapek sucrose agars Hoariness Clam flesh white None - Oat agars Frog flesh white Fleet mustard None ++ : : ; +: ; ++: ; +++: ; ++++:. Note: : Growth slowness; +: Sparseness; ++: More; +++: Clear flourish; ++++: Flourish. Table 2 Test items 表 2 S24 菌株的生理生化特征 Physiological and biochemical properties of strain S24 Results Test items Results Coagulation of milk Glucose Starch hydrolysis Fructose Production of melanoid pigmen Xylose Growth on cellulose Sucrose Gelatin liquefaction Rhamnose Nitrate deoxidization Arabinose Production of H 2 S Glycerol 3% NaCl Mannitol 5% NaCl Inositol Optimal temperature 28 C Sorbitol : +: ; :. Note: +: Positive; : Negative. Ⅰ, (Streptomyces) 2.4 S24 菌株的 6S rrna 序列分析 6Sf/6Sr S24 6S rrna 600 bp( 2), 6S rrna, 6S rrna 426 bp, GenBank( : FJ59682),, S24, Mega4.0 Neighbor-Joining 6S rrna ( 3),, S24 图 2 S24 菌株 6S rrna 片段 % 琼脂糖凝胶电泳结果 Fig. 2 Electrophoresis result with % agarose gel of 6S rrna Note: : PCR Producetion; 2: DL2000 DNA marker., (Streptomyces albireticuli), 98.6% 2.5 S24 菌株的抗菌谱测定结果 4, S24 (A: ; B: ; C: ; D: ), 8 mm 4 E S24, 32 mm~ 33 mm 2.6 S24 菌株抗菌物质对温度的敏感性 5, S24 00 C 20 min, 40 min, 00 min 8.8%, 0 5 Pa 5 min 3.97%,

4 图 4 S24 菌株及其发酵液对粮食和饲料中主要真菌的抑制效果 Inhibition effect of strain fermentation filtrate of S24 on major epiphyte in food and feed 3 结论与讨论 5 Fig.

5 836 微生物学通报 2009, Vol.36, No.2 Fig. 3 图 3 利用 6S rrna 同源序列和邻接法 (Neighbor-Joining) 构建的系统发育树 Phylogenetic tree based on homologous sequences of 6S rrna and Neighbor-Joining analysis Fig. 4 图 4 S24 菌株及其发酵液对粮食和饲料中主要真菌的抑制效果 Inhibition effect of strain fermentation filtrate of S24 on major epiphyte in food and feed 3 结论与讨论 5 Fig. 5 Heat stability of antifungal active substances : *: 0 5 Pa 5 min. Note: *: 0 5 Pa treatment 5 min. 2.7 S24 菌株抗菌物质粗提物对黄曲霉的 MIC 和 MFC RPMI-640, S24, MIC 9.53 µg/ml,, / 0~, 00 µl, PDA, 28 C 48 h, S24 MFC µg/ml,,,, S24, S24, (Streptomyces albireticuli), 6S rrna,, 98.6%,, S24,, DNA-DNA,,, 32 mm~33 mm, ; 96 S24 MIC MFC

6 : S µg/ml µg/ml,,,, [7] S24,, 9500 µg/ml, S24, 参考文献 [] Bluma RV, Etcheverry MG. Influence of Bacillus spp. isolated from maize agroecosystemon growth and aflatoxin B production by Aspergillus section Flavi. Pest Management Science, 2006, 62: [2] Ricci RM, Evans JS, Merrert JJ, et al. Primary cutaneous Aspergillus ustus infection second report case. Journal of the American Academy of Dermatology, 998, 38: [3] Van Burik JA, Colven R, Spaeh DH. Cutaneous aspergillosis. Journal of Clinical Microbiology, 998, 36: [4] Zhang Q, Li L, Zhu M, et al. Primary cutaneous aspergillosis due to Aspergillus flavus: a case report. Chinese Medieal Journal, 2005, 8(3): [5] Oakley EJ, Schmitt F, Jost JP, et al. Some naturally occurring substances: food items and constituents, heterocyclic aromatic amines and mycotoxins. IARC Scientific Publication, 993, 56(6): [6],,,.., 2008, 27(3): [7] Vargas-Arispuro I, Reyes-Baez R, Rivera-Castaieda G, et al. Antifungal lignans from the creosotebush (Larrea tridentata). Industrial Crops and Products, 2005, 22: [8] Sanchez E, Heredia N, Garcia IS. Inhibition of growth and mycotoxin production of Aspergillus flavus and Aspergillus parasiticus by extracts of Agave species. International Journal of Food Microbiology, 2005, 98: [9] Klich MA. Soil fungi of some low-alititude desert cotton fields and ablility of their extracts to inhibit Aspergillus flavus. Mycopathologia, 2003, 42: [0] Yadav V, Gupta J, Mandhan R, et al. Investigations on anti-aspergillus properties of bacterial products. Letters in Applied Microbiology, 2005, 4(4): [],,,. B-FS06., 2007, 23(2): [2] Roy U, Batish VK, Grover S. Production of antifungal substance by Lactococcus lactis subsp. lactischd International Journal of Food Microbiology, 996, 32: [3].. :, 975. [4],.. :, 2004, pp [5] Carl W, Gabriela S. PCR Primer: A Laboratory Manual. Beijing: Chemical Industry Press, [6] Weisburg WG, Barns SM, Pelletier DA, et al. 6S ribosomal DNA amplification for phylogenetic study. J Bacteriol, 99, 73: [7] Klich MA, Lax AR, Bland JM, et al. Influence of iturin A on mycelial weight and aflatoxin production by aspergilles parasiticus in shake culture. Mycopathologia, 993, 23: 稿件书写规范论文中阿拉伯数字的使用,,, 9,,, :

L 8 0 g L ~0 700 g L % 6 00% 1 50% 1 3 NaCl 0 50% NH 4 2 SO % KH 2 PO % CaCO % 0 03% ~ 0 04% 2 24 h 0 02

L 8 0 g L ~0 700 g L % 6 00% 1 50% 1 3 NaCl 0 50% NH 4 2 SO % KH 2 PO % CaCO % 0 03% ~ 0 04% 2 24 h 0 02 38 5 Vol 38 No 5 2014 9 Journal of Jiangxi Normal UniversityNatural Science Sep 2014 1000-5862201405-0501-05 1 1* 2 2 1 3300222 330052 Streptomyces lincolnensis 13 8 6% 12 4% Q 93 A 0 1 2 Streptomyces